The collecting tubule mediates active Na+ resorption, K+ secretion and H+/HCO3 exchange. Each of these processes is driven by ion pumps. The Na, K-ATPase, a member of the E1-E2 class of ion transporting ATPase, is responsible for generating the transepithelial gradients involved in principal cell-mediated ion fluxes. Another member of the E1-E2 family appears to participate in the acid-base transport carried out by intercalated cells. The renal H,K-ATPase has been shown to catalyze electroneutral K+ resorption and proton secretion in perfused collecting tubules. The activities of both of these pumps appear to be governed by several physiologic stimuli. Sodium pump function is modulated by aldosterone and alterations in [Nai]. The renal H,K-ATPase is up- regulated in response to K+ depletion and acidemia. Little is known of the mechanisms through which these influences exert their effects. Recent evidence indicates that the activities of theses pumps are controlled at both the transcriptional and post-transnational levels. Principal cells appear to harbor pools of Na,K-APTase which are maintained in a quiescent state and which are available for rapid mobilization. The renal H,K,-ATPase may be sequestered in an intracellular compartment which can be induced to fuse with the apical plasmalemma. In order to understand the cellular adaptions responsible for such responses, it is necessary to establish the cell biologic and physiologic attributes of these pumps in the context of the cells of the collecting tubule. While much has been learned about the biogenesis of ion pump activity in cultured cell lines, the degree to which these observations are applicable to the highly differentiated cells of the collecting tubule has yet to be established. Furthermore, studies on tissue culture cells have, to date, been unable to shed light on the parameters governing collecting tubule-specific regulatory phenomena. We have developed techniques and probes that will allow us to study the properties of ion pumps and ion pump regulation in physiologically relevant settings. The experiments outlined in this application are designed to 1) establish the cell biologic properties of the Na,K-ATPase in acutely isolated collecting tubules and in a principal cell culture system; 2) isolated a cDNA encoding the renal cells and in situ; and 3) examine the short and long term regulation of the Na,K-ATPase and the renal H,K-ATPase in acutely isolated collecting tubules and in the principal cell culture system. An understanding of the cellular and molecular mechanisms involved in governing the function of the collecting tubules's E1-E2 ion pumps will provide valuable insights into a fascinating and important physiologic system.